Evaluation of the Performance of ACR TI-RADS Also Considering Those Nodules with No Indication of FNAC: A Single-Center Experience.

BACKGROUND: Several US risk stratification score systems (RSSs) have been developed to standardize a thyroid nodule risk of malignancy. It is still a matter of debate which RSS is the most reliable. The purpose of this study is to evaluate: (1) the concordance between the American College of Radiology TI-RADS (ACR TI-RADS) and fine needle aspiration cytology (FNAC), (2) the cancer rate in the ACR TI-RADS categories, (3) the characteristics of nodules evaluated by FNAC even if not formally indicated according to ACR TI-RADS ('not indicated FNACs"). METHODS: From January 2021 to September 2022, patients attending the Endocrinology Unit of the CTO Hospital of Rome for evaluation of thyroid nodules were included. RESULTS: 830 nodules had negative cytology, belonging to TIR2 and TIR1C. One hundred and thirteen nodules were determined to be suspicious for or consistent with malignancy belonging to TIR3B/TIR4/TIR5. Of this last group, 94% were classified as TR4/TR5 nodules. In total, 87/113 underwent surgery. Among these, 73 had histologically proven cancer, 14 turned out to be benign. "Not indicated FNACs" was 623. Among these, 42 cancers were present. CONCLUSIONS: This study confirmed the diagnostic power of ACR TI-RADS. In addition, these data suggest revising the ACR TI-RADS indication to FNAC, especially for TR4.

MAPK activation drives male and female mouse teratocarcinomas from late primordial germ cells.

Germ cell tumors (GCTs) are rare tumors that can develop in both sexes, peaking in adolescents. To understand the mechanisms that underlie germ cell transformation, we established a GCT mouse model carrying a germ-cell-specific BRafV600E mutation with or without heterozygous Pten deletion. Both male and female mice developed monolateral teratocarcinomas containing embryonal carcinoma (EC) cells that showed an aggressive phenotype and metastatic ability. Germ cell transformation started in fetal gonads and progressed after birth leading to gonadal invasion. Early postnatal testes showed foci of tumor transformation, whereas ovaries showed increased number of follicles, multi-ovular follicles (MOFs) and scattered metaphase I oocytes containing follicles. Our results indicate that MAPK (herein referring to Erk1/2) overactivation in fetal germ cells of both sexes can expand their proliferative window leading to neoplastic transformation and metastatic behavior.

Regulation of PDE5 expression in human aorta and thoracic aortic aneurysms.

Aneurysms and dissections affecting thoracic aorta are associated with smooth muscle cell (SMC) dysfunction. NO/cGMP signaling pathway in smooth muscle cells has been shown to be affected in sporadic thoracic aortic aneurysms. We analyzed the mRNA levels of PDE5, a cGMP-hydrolyzing enzyme highly expressed in aortic SMCs, that regulates arterious vascular tone by lowering cGMP levels. We found that aortic tissue obtained from Marfan, tricuspid and bicuspid thoracic aneurysms expressed lower levels of PDE5 mRNA compared to control aortas. In particular, we found that affected aortas showed lower levels of all the PDE5A isoforms, compared to control aortas. Transfection of vascular SMCs (VSMCs) with NOTCH3 activated domain (NICD3) induced the expression of PDE5A1 and A3 protein isoforms, but not that of the corresponding mRNAs. VSMC stimulation with GSNO, a nitric oxide analogue or with 8-br-cGMP, but not with 8-br-cAMP, up-regulated PDE5 and NOTCH-3 protein levels, indicating a negative feedback loop to protect the arterial wall from excessive relaxation. Finally, we found that PDE5 is expressed early during human aorta development, suggesting that if loss of function mutations of PDE5 occur, they might potentially affect aortic wall development.

Histological Study of Discoid Lateral Meniscus in Children and Adolescents: Morphogenetic Considerations.

Background Discoid lateral meniscus is the most frequent variant of the meniscus. Few studies have focused on the histology of discoid menisci. The aim of the present study was to report the histological findings of discoid lateral meniscus in children and adolescents, after arthroscopic partial resection, to give a possible explanation of its developmental etiology. Methods Five patients aged 9, 10, 13, 15, and 17 years were operated on for a 1-piece excision of a discoid lateral meniscus, and the specimens were histologically examined. Results The extracellular matrix showed a different distribution and characteristics depending on the different side of the meniscus. Irregularly oriented collagen fibers in discoid lateral meniscus were found. Cells of different shapes were observed depending on the surficial or deep location in the tissue. There were no blood vessels in the inner part of discoid lateral meniscus. Conclusion The findings of the current study seem to confirm that discoid lateral meniscus arises from variant morphogenesis. Furthermore, the altered distribution and shape of the cells and disorganization of collagen fibers (irrespectively of the age of the patients) may predispose discoid lateral meniscus to degeneration, damage, and tear in young patients also. Level of Evidence Level of evidence 4 (case series).

Discoid lateral meniscus in children and adolescents: a histological study.

BACKGROUND: Discoid lateral meniscus is the most frequent variant of the meniscus. Although the histology of normal menisci in children and in adults has been well described, few studies have focused on the histology of discoid menisci. Furthermore, most of the patients in those studies were adults. The aim of the present study was to report the histological findings of discoid lateral meniscus in a group of children and adolescents, aged between 9 and 18, after arthroscopic partial resection, focusing on cellularity, arrangement of collagen fibers, and vascularity of the excised fragments. Furthermore, to report on MRI findings compared to the histological findings in the same region. METHODS: Six patients (one female and five males) aged 9, 10, 13, 15, 17, and 18, were arthroscopically operated on partial meniscectomy (saucerization) of a discoid lateral meniscus, and the specimens were histologically examined. RESULTS: The extracellular matrix showed a different distribution and characteristics depending on the different side of the meniscus. Irregularly oriented collagen fibers in discoid lateral meniscus were found. There were no blood vessels in the inner part of discoid lateral meniscus in all patients but the 18-year old (in which we observed also endothelials cells, edematous tissue and leaking of erythrocytes in the extracellular matrix). In the discoid lateral menisci analyzed, irregularly oriented collagen fibers with blood vessels were found only in the presence of degenerating tissue. CONCLUSIONS: Discoid lateral meniscus is different from a normal meniscus in terms of vascularity and disorganization of collagen fibers.

Effect Of Microgravity On Aromatase Expression In Sertoli Cells.

Cytochrome P450-aromatase catalyzes estrogen biosynthesis from C19 steroids. In the testis, Sertoli cells express P450-aromatase and represent the primary source of estrogen during prepuberal age. This study focused on the effect of simulated microgravity (SM) on aromatase expression in primary mouse Sertoli cells. When cultured in Rotary Cell Culture System (RCCS), Sertoli cells, formed multicellular three dimensional spheroids (3D). Biological properties were first analyzed in terms of viability, cell cycle, expression of cytoskeletal components and growth factors in comparison to Sertoli cells cultured in spheroids at unit gravity (G). SM did not affect cell viability and proliferation, nor expression of the main cytoskeleton proteins and of growth factors like Kit Ligand (KL) and glial derived neurotrophic factor (GDNF). On the other hand, SM caused a strong increase in P450 aromatase mRNA and protein expression. Interestingly, P450-aromatase was no more inducible by 8-Br-cAMP. The presence of a functional aromatase was confirmed by enrichment of 17ß-estradiol released in the medium by androgen precursors. We concluded that SM causes a significant upregulation of aromatase gene expression in Sertoli cells, leading to a consequent increase in 17ß-estradiol secretion. High level of 17ß-estradiol in the testis could have potentially adverse effects on male fertility and testicular cancer.

Ischemic necrosis of the femoral head: an experimental rabbit model.

To describe the morphology of the proximal femoral epiphysis in a rabbit model from the ischemic insult to the end of the revascularization process. Ischemia of the femoral head was induced in 32 rabbits at the 8th day of life, by sectioning the joint capsule and the ligamentum teres and dislocating the femoral head. Rabbits were sacrificed at 4, 8, 12, 18, 21, 26, 34, and 48 days after surgery and femoral heads were observed histologically. During the first days following the ischemic injury, large areas underwent necrotic changes. Both epiphyseal and physeal cartilage were thicker than normal and less trabecular bone formation was evident. Bone marrow was also diffusely necrotic within the secondary center of ossification. After day 12th, reparative process started with formation of extensive areas of fibrocartilage and several secondary centers of ossifications. At that stage femoral head deformity was already evident. In the following days the secondary centers of ossification cohalesced and epiphyseal and physeal cartilage resumed a normal appearance, but the femur showed a permanent deformity. In newborn rabbits, the ischemic injury to the femoral head blocked the ossification of the epiphyseal and physeal cartilage associated to necrotic bone marrow within the secondary center of ossification of the femoral head as well as to extensive areas of necrosis of epiphyseal and physeal cartilage. Extensive areas of fibrocartilage and small newly formed ossification centers within the femoral epiphysis were the results of the revascularization process, and femoral head deformity became stable afterward.

The transcription factor FBI-1 inhibits SAM68-mediated BCL-X alternative splicing and apoptosis.

Alternative splicing (AS) is tightly coupled to transcription for the majority of human genes. However, how these two processes are linked is not well understood. Here, we unveil a direct role for the transcription factor FBI-1 in the regulation of AS. FBI-1 interacts with the splicing factor SAM68 and reduces its binding to BCL-X mRNA. This, in turn, results in the selection of the proximal 5' splice site in BCL-X exon 2, thereby favoring the anti-apoptotic BCL-XL variant and counteracting SAM68-mediated apoptosis. Conversely, depletion of FBI-1, or expression of a SAM68 mutant lacking the FBI-1 binding region, restores the ability of SAM68 to induce BCL-XS splicing and apoptosis. FBI-1's role in splicing requires the activity of histone deacetylases, whose pharmacological inhibition recapitulates the effects of FBI-1 knockdown. Our study reveals an unexpected function for FBI-1 in splicing modulation with a direct impact on cell survival.

Essential role of Sox2 for the establishment and maintenance of the germ cell line.

Sox2 is a pluripotency-conferring gene expressed in primordial germ cells (PGCs) and postnatal oocytes, but the role it plays during germ cell development and early embryogenesis is unknown. Since Sox2 ablation causes early embryonic lethality shortly after blastocyst implantation, we generated mice bearing Sox2-conditional deletion in germ cells at different stages of their development through the Cre/loxP recombination system. Embryos lacking Sox2 in PGCs show a dramatic decrease of germ cell numbers at the time of their specification. At later stages, we found that Sox2 is strictly required for PGC proliferation. On the contrary, Sox2 deletion in meiotic oocytes does not impair postnatal oogenesis and early embryogenesis, indicating that it is not essential for oocyte maturation or for zygotic development. We also show that Sox2 regulates Kit expression in PGCs and binds to discrete transcriptional regulatory sequences of this gene, which is known to be important for PGCs survival and proliferation. Sox2 also stimulates the expression of Zfp148, which is required for normal development of fetal germ cells, and Rif1, a potential regulator of PGC pluripotency.

The ontogenetic expression pattern of type 5 phosphodiesterase correlates with androgen receptor expression in rat corpora cavernosa.

INTRODUCTION: The mechanisms controlling erection in animals and in humans are mainly age-dependent. However, the ontogenesis of the biochemical machinery of erection is largely unknown. AIM: The aim of this article was to study the expression pattern of androgen receptor (AR) and the major cyclic guanosine monophosphate-hydrolyzing enzyme present in the corpora cavernosa, type 5 phosphodiesterase (PDE5), in the rat penis during development. METHODS: AR and PDE5 expression was tested on ribonucleic acids (RNAs) and proteins extracted from the whole penis or from primary cultures of smooth muscle cells obtained from the corpora cavernosa of 3- (rCC3), 20- (rCC20), and 60- (rCC60) day-old rats. Rat corpus cavernosum cells were characterized by immunocytochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). MAIN OUTCOME MEASURES: Expression of PDE5 and AR messenger RNA (mRNA) and protein have been measured by RT-PCR and Western blot, respectively. RESULTS: A significant increase in PDE5 mRNA expression was observed with RT-PCR from prepuberty to adulthood (0.5 +/- 0.06 vs. 1.6 +/- 0.046 arbitrary units [a.u.]P = 0.049). This age-dependent increase was mirrored by the increase in PDE5 protein expression found when comparing neonatal to adult corpus cavernosum smooth muscle cells (1.5 +/- 0.26 vs. 4.9 +/- 0.59 a.u. P = 0.0038) and the further 1.6-fold increase from rCC20 to rCC60 (4.9 +/- 0.59 vs. 8.0 +/- 0.8 a.u. P = 0.0024). This is the first demonstration of the ontogenetic profile of PDE5 expression in corpus cavernosum smooth muscle. As it has been demonstrated that androgens control PDE5 expression and that PDE5 inhibitors need an optimal androgenic milieu to act perfectly on erection, the expression of AR protein in rat corpus cavernosum cells was then tested by Western blot. A 7.0-fold increase was observed in primary cultured cells from 3 to 60 days old (1.4 +/- 0.38 vs. 9.8 +/- 1.3 a.u. P = 0.0052). CONCLUSION: The increase in ARs during rat penile development parallels that of PDE5 RNA and protein, thus suggesting a positive effect of androgens on PDE5 expression.

The nuclear RNA-binding protein Sam68 translocates to the cytoplasm and associates with the polysomes in mouse spermatocytes.

Translational control plays a crucial role during gametogenesis in organisms as different as worms and mammals. Mouse knockout models have highlighted the essential function of many RNA-binding proteins during spermatogenesis. Herein we have investigated the expression and function during mammalian male meiosis of Sam68, an RNA-binding protein implicated in several aspects of RNA metabolism. Sam68 expression and localization within the cells is stage specific: it is expressed in the nucleus of spermatogonia, it disappears at the onset of meiosis (leptotene/zygotene stages), and it accumulates again in the nucleus of pachytene spermatocytes and round spermatids. During the meiotic divisions, Sam68 translocates to the cytoplasm where it is found associated with the polysomes. Translocation correlates with serine/threonine phosphorylation and it is blocked by inhibitors of the mitogen activated protein kinases ERK1/2 and of the maturation promoting factor cyclinB-cdc2 complex. Both kinases associate with Sam68 in pachytene spermatocytes and phosphorylate the regulatory regions upstream and downstream of the Sam68 RNA-binding motif. Molecular cloning of the mRNAs associated with Sam68 in mouse spermatocytes reveals a subset of genes that might be posttranscriptionally regulated by this RNA-binding protein during spermatogenesis. We also demonstrate that Sam68 shuttles between the nucleus and the cytoplasm in secondary spermatocytes, suggesting that it may promote translation of specific RNA targets during the meiotic divisions.

Meiotic progression of isolated mouse spermatocytes under simulated microgravity.

Progression through the prophase of the first meiotic division can be obtained in culture by treatment of mouse spermatocytes with the serine/threonine phosphatase inhibitor okadaic acid. Chromosome condensation during this G2/M transition involves the activation of the MAPK pathway, which causes the activation of Nek2 and the phosphorylation of the chromatin architectural protein Hmga2. In an effort to set up conditions to allow a spontaneous progression of mouse spermatocytes through meiosis, we have investigated the cell-cycle features of these cells cultured for 24 h with a rotary cell culture system in a humidified atmosphere in a thermostatic incubator to simulate a microgravity environment. Morphological analysis of nuclear squashes indicated a 2-fold increase in late-pachytene spermatocytes with highly condensed chromosomes, and a contemporaneous decrease of mid-pachytene cells with less condensed chromatin. Microgravity induced a 2-fold activation of the cyclinB-cdc2 complex, confirming at the molecular level that cell-cycle progression had occurred. Moreover, using immuno-kinase assays with specific substrates we have demonstrated that the meiotic progression obtained under microgravity conditions is accompanied by activation of the Erk1/p90Rsk2 pathway. These data indicated that activation of the MAPK pathway correlates with chromatin condensation even under conditions in which meiotic progression occurs spontaneously and is not induced by a drug. We suggest that culture under microgravity conditions might help to release the block that inhibits isolated spermatocytes from progressing through prophase at unit gravity, and to study the physiological events of germ cell differentiation in vitro.